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HUMAN ADIPOCYTE FABP ELISA (HUMAN FABP4 ELISA)

Product Data Sheet

Cat. No.: RD191036200R

For Research Use Only

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CONTENTS

1. INTENDED USE 3

2. STORAGE, EXPIRATION 3

3. INTRODUCTION 4

4. TEST PRINCIPLE 5

5. PRECAUTIONS 5

6. TECHNICAL HINTS 6

7. REAGENT SUPPLIED 6

8. MATERIAL REQUIRED BUT NOT SUPPLIED 7

9. PREPARATION OF REAGENTS 7

10. PREPARATION OF SAMPLES 9

11. ASSAY PROCEDURE 10

12. CALCULATIONS 11

13. PERFORMANCE CHARACTERISTICS 12

14. DEFINITION OF THE STANDARD 17

15. PRELIMINARY POPULATION AND CLINICAL DATA 17

16. METHOD COMPARISON 20

17. TROUBLESHOOTING AND FAQS 21

18. REFERENCES 22

19. EXPLANATION OF SYMBOLS 24

This kit is manufactured by: Bio Vendor Research and Diagnostic Products, s.r.o.

Use only the current version of Product Data Sheet enclosed with the kit!

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1. INTENDED USE

The RD191036200R Human AFABP ELISA is a sandwich enzyme immunoassay for the quantitative measurement of human AFABP.

Features

It is intended for research use only

The total assay time is less than 4 hours

The kit measures total AFABP in serum and plasma (EDTA, citrate, heparin)

Assay format is 96 wells

Quality Controls are human serum based

Standard is recombinant protein based

Components of the kit are provided ready to use, concentrated or lyophilized

Patent Application Number: DE 10 2005 034 788.6

2. STORAGE, EXPIRATION

Store the complete kit at 2-8°C. Under these conditions, the kit is stable until the expiration date (see label on the box). For stability of opened reagents see Chapter 9.

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3. INTRODUCTION

Protein definition: Protein name: Adipocyte FABP (AFABP) Synonyms: Fatty acid-binding protein, adipocyte (A-FABP) Adipocyte lipid-binding protein (ALBP) Gene name: FABP4 Swissprot: P15090 NCBI / Protein: P15090 Adipocyte fatty acid binding protein AFABP is a 15 kDa member of the intracellular fatty acid binding protein (FABP) family, which is known for the ability to bind fatty acids and related compounds (bile acids or retinoids) in an internal cavity. AFABP is expressed in a differentiation-dependent fashion in adipocytes and is a critical gene in the regulation of the biological function of these cells. In mice, targeted mutations in FABP4 (mouse gene is also called aP2 and its relevant protein P2 adipocyte protein or 3T3-L1 lipid binding protein) provide significant protection from hyperinsulinemia and insulin resistance in the context of both dietary and genetic obesity. Adipocytes obtained from AFABP-deficient mice also have reduced efficiency of lipolysis in vitro and in vivo, and these mice exhibited moderately improved systemic dyslipidemia. Recent studies also demonstrated AFABP expression in human macrophages upon differentiation and activation. In these cells, AFABP modulates inflammatory responses and cholesterol ester accumulation, and total or macrophage-specific AFABP deficiency confers dramatic protection against atherosclerosis in the apoE-/- mice. These results indicate a central role for AFABP in the development of major components of the metabolic syndrome through its distinct actions in adipocytes and macrophages. Besides being active within the cell, AFABP appears to be a secreted protein (for normal levels and correlations with certain metabolic parameters see chapter 15). The extracellular role of secreted AFABP remains to be determined. Areas of investigation: Energy metabolism and body weight regulation

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4. TEST PRINCIPLE

In the BioVendor Human AFABP ELISA, standards, quality controls and samples are incubated in microplate wells pre-coated with polyclonal anti-human AFABP antibody. After 60 minutes incubation and washing, biotin labelled polyclonal anti-human AFABP antibody is added and incubated for 60 minutes with captured AFABP. After another washing, streptavidin-HRP conjugate is added. After 30 minutes incubation and the last washing step, the remaining conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of AFABP. A standard curve is constructed by plotting absorbance values against concentrations of standards, and concentrations of unknown samples are determined using this standard curve.

5. PRECAUTIONS

For professional use only

Wear gloves and laboratory coats when handling immunodiagnostic materials

Do not drink, eat or smoke in the areas where immunodiagnostic materials are being handled

This kit contains components of human origin. These materials were found non-reactive for HBsAg, HCV antibody and for HIV 1/2 antigen and antibody. However, these materials should be handled as potentially infectious, as no test can guarantee the complete absence of infectious agents

This kit contains components of animal origin. These materials should be handled as potentially infectious

Avoid contact with the acidic Stop Solution and Substrate Solution, which contains hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing protection when handling these reagents. Stop and/or Substrate Solutions may cause skin/eyes irritation. In case of contact with the Stop Solution and the Substrate Solution wash skin/eyes thoroughly with water and seek medical attention, when necessary

The materials must not be pipetted by mouth

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6. TECHNICAL HINTS

Reagents with different lot numbers should not be mixed

Use thoroughly clean glassware

Use deionized (distilled) water, stored in clean containers

Avoid any contamination among samples and reagents. For this purpose, disposable tips should be used for each sample and reagent

Substrate Solution should remain colourless until added to the plate. Keep Substrate Solution protected from light

Stop Solution should remain colourless until added to the plate. The colour developed in the wells will turn from blue to yellow immediately after the addition of the Stop Solution. Wells that are green in colour indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution

Dispose of consumable materials and unused contents in accordance with applicable national regulatory requirements

7. REAGENT SUPPLIED

Kit Components State Quantity

Antibody Coated Microtiter Strips ready to use 96 wells

Biotin Labelled Antibody Conc. (100x) concentrated 0.15 ml

Streptavidin-HRP Conjugate ready to use 13 ml

Master Standard lyophilized 1 vial

Quality Control HIGH lyophilized 1 vial

Quality Control LOW lyophilized 1 vial

Dilution Buffer ready to use 2 x 13 ml

Biotin-Ab Diluent ready to use 13 ml

Wash Solution Conc. (10x) concentrated 100 ml

Substrate Solution ready to use 13 ml

Stop Solution ready to use 13 ml

Product Data Sheet + Certificate of Analysis - 1 pc

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8. MATERIAL REQUIRED BUT NOT SUPPLIED

Deionized (distilled) water

Test tubes for diluting samples

Glassware (graduated cylinder and bottle) for Wash Solution (Dilution Buffer)

Precision pipettes to deliver 10-1000 l with disposable tips

Multichannel pipette to deliver 100 l with disposable tips

Absorbent material (e.g. paper towels) for blotting the microtitrate plate after washing

Vortex mixer

Orbital microplate shaker capable of approximately 300 rpm

Microplate washer (optional). [Manual washing is possible but not preferable.]

Microplate reader with 450 10 nm filter, preferably with reference wavelength 630 nm (alternatively another one from the interval 550-650 nm)

Software package facilitating data generation and analysis (optional)

9. PREPARATION OF REAGENTS

All reagents need to be brought to room temperature prior to use

Always prepare only the appropriate quantity of reagents for your test

Do not use components after the expiration date marked on their label

Assay reagents supplied ready to use: Antibody Coated Microtiter Strips Stability and storage: Return the unused strips to the provided aluminium zip-sealed bag with desicant and seal carefully. Remaining Microtiter Strips are stable 3 months stored at 2-8°C and protected from the moisture.

Streptavidin-HRP Conjugate Dilution Buffer Biotin-Ab Diluent Substrate Solution Stop Solution Stability and storage: Opened reagents are stable 3 months when stored at 2-8°C.

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Assay reagents supplied concentrated or lyophilized: Human AFABP Master Standard Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for reconstitution of standard!!! Reconstitute the lyophilized Master Standard with Dilution Buffer just prior to the assay. Let it dissolve at least 15 minutes with occasional gentle shaking (not to foam). The resulting concentration of the human AFABP in the stock solution is 25 ng/ml. Prepare set of standards using Dilution Buffer as follows:

Volume of Standard Dilution Buffer Concentration

Stock - 25 ng/ml

500 l of stock 750 l 10 ng/ml

500 l of 10 ng/ml 500 l 5 ng/ml

500 l of 5 ng/ml 500 l 2.5 ng/ml

500 l of 2.5 ng/ml 750 l 1 ng/ml

500 l of 1 ng/ml 500 l 0.5 ng/ml

Prepared Standards are ready to use, do not dilute them. Stability and storage: Standard stock solution (25 ng/ml) should be aliquoted and frozen at –20°C for 3 months. Avoid repeated freeze/thaw cycles.

Do not store the diluted Standard solutions.

Quality Controls HIGH, LOW Refer to the Certificate of Analysis for current volume of distilled water needed for reconstitution and for current Quality Control concentration!!! Reconstitute each Quality Control (HIGH and LOW) with distilled water just prior to the assay. Let it dissolve at least 15 minutes with occasional gentle shaking (not to foam).

Dilute reconstituted Quality Controls 10x with Dilution Buffer, e.g. 20 l of Quality Control +

180 l of Dilution Buffer when assaying samples in singlets, or preferably 30 l of Quality

Control + 270 l of Dilution Buffer for duplicates. Stability and storage: The reconstituted Quality Controls must be used immediately or stored frozen at -20°C for 3 months. Avoid repeated freeze/thaw cycles. Do not store the diluted Quality Controls. Note: Concentration of analyte in Quality Controls need not be anyhow associated with normal and/or pathological concentrations in serum or another body fluid. Quality Controls serve just for control that the kit works in accordance with PDS and CoA and that ELISA test was carried out properly.

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Biotin Labelled Antibody Conc. (100x) Prepare the working Biotin Labelled Antibody solution by adding 1 part Biotin Labelled

Antibody Concentrate (100x) with 99 parts Biotin-Ab Diluent. Example: 10 l of Biotin Labelled

Antibody Concentrate (100x) + 990 l of Biotin-Ab Diluent for 1 strip (8 wells). Stability and storage: Opened Biotin Labelled Antibody Concentrate (100x) is stable 3 months when stored at 2-8°C. Do not store the diluted Biotin Labelled Antibody solution. Wash Solution Conc. (10x) Dilute Wash Solution Concentrate (10x) ten-fold in distilled water to prepare a 1x working solution. Example: 100 ml of Wash Solution Concentrate (10x) + 900 ml of distilled water for use of all 96-wells. Stability and storage: The diluted Wash Solution is stable 1 month when stored at 2-8°C. Opened Wash Solution Concentrate (10x) is stable 3 months when stored at 2-8°C.

10. PREPARATION OF SAMPLES

The kit measures AFABP in serum and plasma (EDTA, citrate, heparin) samples.

Samples should be assayed immediately after collection or should be stored at -20°C. Mix thoroughly thawed samples just prior to the assay and avoid repeated freeze/thaw cycles, which may cause erroneous results. Avoid using hemolyzed or lipemic samples.

Dilute serum or plasma samples 10x with Dilution Buffer just prior to the assay, e.g. 20 l of

sample + 180 l of Dilution Buffer for singlets, or preferably 30 l of sample + 270 l of Dilution Buffer for duplicates. Mix well (not to foam). Vortex is recommended. Stability and storage: Samples should be stored at -20°, or preferably at -70°C for long-term storage. Avoid repeated freeze/ thaw cycles. Do not store the diluted samples.

See Chapter 13 for stability of serum and plasma samples when stored at 2-8°C, effect of freezing/thawing and effect of sample matrix (serum/plasma) on the concentration of AFABP. Note: It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results.

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11. ASSAY PROCEDURE

1. Pipet 100 l of diluted Standards, Quality Controls, Dilution Buffer (=Blank) and samples, preferably in duplicates, into the appropriate wells. See Figure 1 for example of work sheet.

2. Incubate the plate at room temperature (ca. 25°C) for 1 hour, shaking at ca. 300 rpm on an orbital microplate shaker.

3. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and tap the plate strongly against paper towel.

4. Add 100 l of Biotin Labelled Antibody into each well. 5. Incubate the plate at room temperature (ca. 25°C) for 1 hour, shaking at ca. 300 rpm on

an orbital microplate shaker. 6. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and

tap the plate strongly against paper towel.

7. Add 100 l of Streptavidin-HRP Conjugate into each well. 8. Incubate the plate at room temperature (ca. 25°C) for 30 minutes, shaking at ca.

300 rpm on an orbital microplate shaker. 9. Wash the wells 5-times with Wash Solution (0.35 ml per well). After final wash, invert and

tap the plate strongly against paper towel.

10. Add 100 l of Substrate Solution into each well. Avoid exposing the microtiter plate to direct sunlight. Covering the plate with e.g. aluminium foil is recommended.

11. Incubate the plate for 10 minutes at room temperature. The incubation time may be extended [up to 20 minutes] if the reaction temperature is below than 20°C. Do not shake the plate during the incubation.

12. Stop the colour development by adding 100 l of Stop Solution. 13. Determine the absorbance of each well using a microplate reader set to 450 nm, preferably with the reference wavelength set to 630 nm (acceptable range: 550-650 nm). Subtract readings at 630 nm (550-650 nm) from the readings at 450 nm. The absorbance should be read within 5 minutes following step 12. Note: If the microplate reader is not capable of reading absorbance greater than the absorbance of the highest standard, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine AFABP concentration of off-scale samples. The readings at 405 nm should not replace the on-scale readings at 450 nm. Note 2: Manual washing: Aspirate wells and pipet 0.35 ml Wash Solution into each well. Aspirate wells and repeat twice. After final wash, invert and tap the plate strongly against paper towel. Make certain that Wash Solution has been removed entirely.

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Figure 1: Example of a work sheet.

12. CALCULATIONS

Most microplate readers perform automatic calculations of analyte concentration. The Standard curve is constructed by plotting the mean absorbance (Y) of Standards against log of the known concentration (X) of Standards, using the four-parameter algorithm. Results are reported as concentration of AFABP ng/ml in samples. Alternatively, the logit log function can be used to linearize the standard curve (i.e. logit of the mean absorbance (Y) is plotted against log of the known concentration (X) of Standards. The measured concentration of samples and Quality Controls calculated from the standard curve must be multiplied by their respective dilution factor, because samples and Quality Controls have been diluted prior to the assay. e.g. 1.57 ng/ml (from standard curve) x 10 (dilution factor) = 15.7 ng/ml.

strip 1+2 strip 3+4 strip 5+6 strip 7+8 strip 9+10 strip 11+12

A Standard 25 QC HIGH Sample 8 Sample 16 Sample 24 Sample 32

B Standard 10 Sample 1 Sample 9 Sample 17 Sample 25 Sample 33

C Standard 5 Sample 2 Sample 10 Sample 18 Sample 26 Sample 34

D Standard 2.5 Sample 3 Sample 11 Sample 19 Sample 27 Sample 35

E Standard 1 Sample 4 Sample 12 Sample 20 Sample 28 Sample 36

F Standard 0.5 Sample 5 Sample 13 Sample 21 Sample 29 Sample 37

G Blank Sample 6 Sample 14 Sample 22 Sample 30 Sample 38

H QC LOW Sample 7 Sample 15 Sample 23 Sample 31 Sample 39

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Human AFABP ELISA

Standard Curve

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

0 1 10 100

Concentration of Hu AFABP (ng/ml)

Abs

orba

nce

(A45

0 nm

-A63

0 nm

)

Figure 2: Typical Standard Curve for Human AFABP ELISA.

13. PERFORMANCE CHARACTERISTICS

Typical analytical data of BioVendor Human AFABP ELISA are presented in this chapter.

Sensitivity Limit of Detection (LOD) (defined as concentration of analyte giving absorbance higher than mean absorbance of blank* plus three standard deviations of the absorbance of blank: Ablank + 3xSDblank) is calculated from the real AFABP values in wells and is 0.05 ng/ml. *Dilution Buffer is pipetted into blank wells.

Limit of assay Results exceeding AFABP level of 25 ng/ml should be repeated with more diluted samples. Dilution factor needs to be taken into consideration in calculating the AFABP concentration.

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Specificity The antibodies used in this ELISA are specific for human AFABP. Sera of several mammalian species were measured in the assay. See results below. For details please contact us at info@biovendor.com.

Mammalian serum sample

Observed crossreactivity

Bovine no

Cat no

Dog yes

Goat no

Hamster no

Horse no

Monkey yes

Mouse yes

Pig no

Rabbit no

Rat no

Sheep no

Presented results are multiplied by respective dilution factor

Precision Intra-assay (Within-Run) (n=8)

Sample Mean (ng/ml)

SD (ng/ml)

CV (%)

1 9.08 0.21 2.31

2 22.75 0.62 2.72

Inter-assay (Run-to-Run) (n=6)

Sample Mean (ng/ml)

SD (ng/ml)

CV (%)

1 31.88 1.74 5.46

2 53.13 1.24 2.34

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Spiking Recovery Serum samples were spiked with different amounts of human AFABP and assayed.

Sample Observed (ng/ml)

Expected (ng/ml)

Recovery O/E (%)

1 18.63 69.43 45.31 31.87

- 68.63 43.63 28.63

- 101.2 103.9 111.3

2 23.44 69.84 52.55 35.95

- 73.44 48.44 33.44

- 95.1 108.5 107.5

Linearity Serum samples were serially diluted with Dilution Buffer and assayed.

Sample Dilution Observed (ng/ml)

Expected (ng/ml)

Recovery O/E (%)

1 - 2x 4x 8x

59.50 29.90 14.41 7.57

- 29.75 14.87 7.44

- 100.5 96.8 101.7

2 - 2x 4x 8x

52.62 26.37 13.01 6.77

- 26,31 13.15 6.58

- 100.2 98.9 102.9

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Effect of sample matrix Citrate, heparin and EDTA plasmas were compared to respective serum samples from the same 10 individuals. Results are shown below:

Volunteer No.

Serum (ng/ml)

Plasma (ng/ml)

EDTA Citrate Heparin

1 6.87 6.05 4.86 6.41

2 13.51 12.18 10.93 12.16

3 14.94 13.28 11.71 9.74

4 12.67 12.04 11.30 12.55

5 20.33 14.53 13.82 17.72

6 10.46 9.43 8.94 9.68

7 16.09 14.21 12.30 12.36

8 13.94 12.30 11.90 13.94

9 14.31 12.90 10.3 13.80

10 7.55 6.92 6.64 7.42

Mean (ng/ml) 13.07 11.38 10.33 11.58

Mean Plasma/Serum (%)

- 87.1 79.1 88.6

Coefficient of determination R2

- 0.91 0.84 0.91

Effect of Sample Matrix

0

5

10

15

20

25

1 2 3 4 5 6 7 8 9 10

Volunteers

Con

cent

ratio

n of

AF

AB

P (

ng/m

l)

Serum

EDTA Plasma

Citrate Plasma

Heparin Plasma

Figure 3: AFABP levels measured using Human AFABP ELISA from 10 individuals using serum, EDTA, citrate and heparin plasma, respectively.

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Stability of samples stored at 2-8°C Samples should be stored at –20°C. However, no decline in concentration of AFABP was observed in serum and plasma samples after 7 days when stored at 2-8°C. To avoid microbial

contamination, samples were treated with -aminocaproic acid and thimerosal, resulting in the final concentration of 0.03% and 0.05%, respectively.

Sample Incubation

Temp, Period Serum (ng/ml)

Plasma (ng/ml)

EDTA Citrate Heparin

1 -20°C 14.01 13.22 12.10 14.35

2-8°C, 1 day 13.93 12.75 10.88 13.48 2-8°C, 7 days 14.05 12.98 11.12 13.44

2 -20°C 5.40 3.96 3.84 5.67

2-8°C, 1 day 5.78 4.54 3.72 5.35 2-8°C, 7 days 5.18 4.47 3.24 4.31

3 -20°C 14.07 11.49 11.66 14.03

2-8°C, 1 day 14.21 11.68 11.49 12.63 2-8°C, 7 days 13.56 11.59 11.43 12.73

Effect of Freezing/Thawing No decline was observed in concentration of human AFABP in serum and plasma samples after repeated (5x) freeze/thaw cycles. However it is recommended to avoid unnecessary repeated freezing/thawing of the samples.

Sample Number of f/t

cycles Serum (ng/ml)

Plasma (ng/ml)

EDTA Citrate Heparin

1 1x 13.72 12.45 11.83 13.23 3x 12.96 12.71 10.22 13.27 5x 11.71 12.06 11.57 12.37

2 1x 18.87 16.00 15.35 17.77

3x 18.91 18.55 14.96 17.50 5x 19.57 16.59 14.67 15.79

3 1x 13.59 9.43 9.68 10.95 3x 11.71 10.37 8.22 11.09 5x 11.67 10.62 10.78 10.86

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14. DEFINITION OF THE STANDARD

The recombinant protein is used as the standard in this assay. The recombinant AFABP is a 14.7 kDa protein containing 132 amino acid residues. Master Standard used in this kit contains 25 ng of AFABP measured by BCA method (Sigma-Aldrich).

15. PRELIMINARY POPULATION AND CLINICAL DATA

In our preliminary study, we investigated relations between serum AFABP value and some basic population parameters, parameters of lipid metabolism and parameters of insulin sensitivity.

Normal value and normal range in human serum The mean value study with sera samples from 66 random selected women, 35-52 years old, has been established with the Biovendor Human AFABP ELISA in our laboratory (n=66, mean ± SEM): 19.58 ± 1.01 ng/ml. The normal range with sera samples from 66 random selected women, 35-52 years old, has been established with the Biovendor Human AFABP ELISA in our laboratory: Normal range (mean ± 2 SD): 19.58 ± 16.32 ng/ml.

Reference range The data quoted in these instructions should be used for guidance only. It is recommended that each laboratory include its own panel of control sample in the assay. Each laboratory should establish its own normal and pathological references ranges for AFABP levels with the assay.

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Histogram of serum AFABP values, women

35-52 years old, N=66

0

5

10

15

20

25

0.0

5.0

11.0

17.0

23.0

29.0

35.0

41.0

47.0

53.0

59.0

65.0

71.0

77.0

83.0

89.0

95.0

101.

0

Concentration of AFABP (ng/ml)

Fre

quen

cy

Figure 4: Histogram of serum AFABP values shows Gaussian distribution.

Influence of gender on AFABP value in human serum The comparison of serum AFABP values between men (n=10) and women (n=26) selected by age (35-52 years old volunteers) did not show any significant difference. Results are presented as mean values:

Age (years)

Total cholesterol

(mmol/l)

HDL (mmol/l)

LDL (mmol/l)

Triglycerides (mmol/l)

AFABP (mmol/l)

Women 45.5 5.21 1.46 3.35 1.13 21.28

Men 43.7 5.08 1.06 3.70 1.61 21.44

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Clinical relevance of serum AFABP value We have found correlations between serum AFABP value and triglycerides, HDL and LDL values.

0

20

40

60

80

100

120

140

0 1 2 3 4 5 6

Concentration of Triglycerides (mmol/l)

Con

cent

ratio

n of

AF

AB

P (

ng/m

l)

Figure 5: Serum AFABP positively correlates with triglycerides.

0

20

40

60

80

100

120

140

0 0.5 1 1.5 2 2.5 3 3.5

Concentration of HDL cholesterol (mmol/l)

Con

cent

ratio

n of

AF

AB

P (

ng/m

l)

Figure 6: Serum AFABP negatively correlates with HDL.

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0

20

40

60

80

100

120

140

0 1 2 3 4 5 6 7 8

Concentration of LDL cholesterol (mmol/l)

Con

cent

raci

on o

f AF

AB

P (

ng/m

l)

Figure 7: Serum AFABP positively correlates with LDL. Increased serum AFABP values were found in obese volunteers in a similar study. AFABP values are presented as mean +/- SD:

Volunteer No. (F/M)

Age (years) AFABP (ng/ml)

Obese (BMI > 25)

129 (62/67) 53.9 ± 12.7 32.3 ± 14.8

Non-obese (BMI < 25)

100 (46/54) 57.6 ± 12.8 20.0 ± 9.8

16. METHOD COMPARISON

The BioVendor Human AFABP ELISA was not compared to the other commercial immunoassays.

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17. TROUBLESHOOTING AND FAQS

Weak signal in all wells Possible explanations:

Omission of a reagent or a step

Improper preparation or storage of a reagent

Assay performed before reagents were allowed to come to room temperature

Improper wavelength when reading absorbance

High signal and background in all wells Possible explanations:

Improper or inadequate washing

Overdeveloping; incubation time with Substrate Solution should be decreased before addition of Stop Solution

Incubation temperature over 30°C

High coefficient of variation (CV) Possible explanation:

Improper or inadequate washing

Improper mixing Standards, Quality Controls or samples

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18. REFERENCES

References to adipocyte FABP:

Makowski L, Brittingham KC, Reynolds JM, Suttles J and Hotamisligil GS: The Fatty Acid-binding Protein, aP2, Coordinates Macrophage Cholesterol Trafficking and Inflammatory Activity. J Biol Chem. Apr 1; 280(13): 12888-12895 (2005)

Maeda K, Cao H, Kono K, Gorgun CZ, Furuhashi M, Uysal KT, Cao Q, AtsumiG, Malone H, Krishnan B, Minokoshi Y, Kahn BB, Parker RA and Hotamisligil GS: Adipocyte/macrophage fatty acid binding proteins control integrated metabolic responses in obesity and diabetes. Cell Metabolism. February; 1(2): 107-119 (2005)

Boord JB, Maeda K, Makowski L, Babaev VR, Fazio S, Linton MF, Hotamisligil GS: Combined adipocyte-macrophage fatty acid-binding protein deficiency improves metabolism, atherosclerosis, and survival in apolipoprotein E-deficient mice. Circulation. Sep 14; 110(11): 1492-1498 (2004)

Lehmann F, Haile S, Axen E, Medina C, Uppenberg J, Svensson S, Lundback T, Rondahl L, Barf T: Discovery of inhibitors of human adipocyte fatty acid-binding protein, a potential type 2 diabetes target. Bioorg Med Chem Lett. Sep 6; 14(17): 4445-4448 (2004)

Damcott CM, Moffett SP, Feingold E, Barmada MM, Marshall JA, Hamman RF, Ferrell RE: Genetic variation in fatty acid-binding protein-4 and peroxisome proliferator-activated receptor gamma interactively influence insulin sensitivity and body composition in males. Metabolism. Mar; 53(3): 303-9 (2004)

Jenkins-Kruchten AE, Bennaars-Eiden A, Ross JR, Shen WJ, Kraemer FB, Bernlohr DA: Fatty acid-binding protein-hormone-sensitive lipase interaction. Fatty acid dependence on binding.J Biol Chem. Nov 28; 278(48): 47636-47643 (2003)

Hertzel AV, Bennaars-Eiden A, Bernlohr DA: Increased lipolysis in transgenic animals overexpressing the epithelial fatty acid binding protein in adipose cells. J Lipid Res. Dec; 43(12): 2105-2111 (2002)

Fu Y, Luo N, Lopes-Virella MF, Garvey WT: The adipocyte lipid binding protein (ALBP/aP2) gene facilitates foam cell formation in human THP-1 macrophages. Atherosclerosis. Dec; 165(2): 259-269 (2002)

Fisher RM, Hoffstedt J, Hotamisligil GS, Thorne A, Ryden M: Effects of obesity and weight loss on the expression of proteins involved in fatty acid metabolism in human adipose tissue. Int J Obes Relat Metab Disord. Oct; 26(10): 1379-1385 (2002)

Boord JB, Maeda K, Makowski L, Babaev VR, Fazio S, Linton MF, Hotamisligil GS: Adipocyte fatty acid-binding protein, aP2, alters late atherosclerotic lesion formation in severe hypercholesterolemia. Arterioscler Thromb Vasc Biol. Oct 1; 22(10): 1686-1691(2002)

Scheja L, Makowski L, Uysal KT, Wiesbrock SM, Shimshek DR, Meyers DS, Morgan M, Parker RA, Hotamisligil GS: Altered insulin secretion associated with reduced lipolytic efficiency in aP2-/- mice. Diabetes. Oct; 48(10): 1987-1994 (1999)

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References to this product:

Milner KL, van der Poorten D, Xu A, Bugianesi E, Kench JG, Lam KS, Chisholm DJ, George J. Adipocyte fatty acid binding protein levels relate to inflammation and fibrosis in nonalcoholic fatty liver disease. Hepatology. Jun; 49(6):1926-34 (2009)

Yeung DC, Xu A, Tso AW, Chow WS, Wat NM, Fong CH, Tam S, Sham PC, Lam KS. Circulating levels of adipocyte and epidermal fatty acid-binding proteins in relation to nephropathy staging and macrovascular complications in type 2 diabetic patients. Diabetes Care. Jan; 32(1):132-4 (2009)

Stejskal D, Karpisek M, Bronsky J. Serum adipocyte-fatty acid binding protein discriminates patients with permanent and temporary body weight loss. J Clin Lab Anal.; 22(5):380-2 (2008)

Karpisek M, Stejskal D, Kotolova H, Kollar P, Janoutova G, Ochmanova R, Cizek L, Horakova D, Yahia RB, Lichnovska R, Janout V. Treatment with atorvastatin reduces serum adipocyte-fatty acid binding protein value in patients with hyperlipidaemia. Eur J Clin Invest. Aug; 37(8):637-42 (2007)

Tso AW, Xu A, Sham PC, Wat NM, Wang Y, Fong CH, Cheung BM, Janus ED, Lam KS. Serum adipocyte fatty acid binding protein as a new biomarker predicting the development of type 2 diabetes: a 10-year prospective study in a Chinese cohort. Diabetes Care. Oct; 30(10):2667-72 (2007)

Yeung DC, Xu A, Cheung CW, Wat NM, Yau MH, Fong CH, Chau MT, Lam KS. Serum adipocyte fatty acid-binding protein levels were independently associated with carotid atherosclerosis. Arterioscler Thromb Vasc Biol. Aug; 27(8):1796-802 (2007)

Xu A, Tso AW, Cheung BM, Wang Y, Wat NM, Fong CH, Yeung DC, Janus ED, Sham PC, Lam KS: Circulating adipocyte-fatty acid binding protein levels predict the development of the metabolic syndrome: a 5-year prospective study. Circulation. Mar 27; 115(12): 1537-1543 (2007)

Bronsky J, Karpisek M, Bronska E, Pechova M, Jancikova B, Kotolova H, Stejskal D, Prusa R, Nevoral J: Adiponectin, adipocyte fatty acid binding protein, and epidermal fatty acid binding protein: Proteins newly identified in human breast milk. Clin Chem. Sep; 52(9): 1763-1770 (2006)

von Eynatten M, Schneider JG, Allolio B: Adipocyte fatty acid binding protein (A-FABP) ist ein neuer Serum-Marker für Adipositas, Insulinresistenz und Metabolisches Syndrom. Diabetologie. 1: S23-S172 (2006)

Xu A, Wang Y, Xu JY, Stejskal D, Tam S, Zhang J, Wat NM, Wong WK, Lam KS: Adipocyte fatty acid–binding protein is a plasma biomarker closely associated with obesity and metabolic syndrome. Clin Chem. Mar; 52(3): 405-413 (2006)

Stejskal D and Karpisek M: Adipocyte fatty acid binding protein (A-FABP) in Caucasian population: a new marker of metabolic syndrome? Eur J Clin Invest. Sep; 36(9): 621-625 (2006)

For more references on this product see our WebPages at www.biovendor.com

Page 24 of 28 ENG.001.A

19. EXPLANATION OF SYMBOLS

Catalogue number

Content

Lot number

See instructions for use

Biological hazard

Expiry date

Storage conditions

Identification of packaging materials

Page 25 of 28 ENG.001.A

Antibody Coated Microtiter Plate

Reconstitute Master Standard and prepare set of standards

Reconstitute QCs and dilute them 10x, dilute samples 10x

Add Standards, QCs and samples

100 l

Incubate at RT for 1 hour/300 rpm

Prepare Wash Solution

Wash 5x

Incubate at RT for 1 hour/300rpm

Incubate at RT for 30 min/300rpm

Incubate at RT for 10 min

Wash 5x

Wash 5x

Add Streptavidin HRP Conjugate

100 l

Add Substrate Solution

100 l

Add Stop Solution

100 l

Read absorbance and calculate results

Assay Procedure Summary

Add Biotin Labelled Antibody Solution

100 l

Prepare Biotin Labeled

Antibody Solution

Page 26 of 28 ENG.001.A

12

11

10

9

8

7

6

5

4

3

2

1

A

B

C

D

E

F

G

H

Page 27 of 28 ENG.001.A

NOTES

Page 28 of 28 ENG.001.A

HEADQUARTERS: BioVendor - Laboratorní medicína a.s.

Karasek 1767/1

621 00 Brno CZECH REPUBLIC

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E-mail: info@biovendor.com sales@biovendor.com Web: www.biovendor.com

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Phone: +1-828-575-9250 +1-800-404-7807 Fax: +1-828-575-9251

E-mail: infoUSA@biovendor.com

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